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Title: In Situ Immune Infrared Fluorescent Staining for Detection and Quantification of Bluetongue Virus in Cullicoides Insect Cell Culture

Author
item Mecham, James
item Brown, Philip
item McHolland, Linda

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/5/2009
Publication Date: 6/1/2009
Citation: Mecham, J.O., Brown, P.L., Mcholland, L.E. 2009. In Situ Immune Infrared Fluorescent Staining for Detection and Quantification of Bluetongue Virus in Cullicoides Insect Cell Culture. Journal of Virological Methods. 158, 110-113.

Interpretive Summary: Bluetongue virus (BTV) infects sheep, cattle and other ruminants and is transmitted by Culicoides spp. of biting midges. In this laboratory, cell lines have been produced from C. sonorensis that have proven to be particularly useful for various studies on BTV and the insect vector. However, since BTV does not produce detectable damage in these cells, indirect methods, such as co-cultivation with susceptible mammalian cells, are necessary for detection and quantifying virus replication in the insect cells. This paper describes the development of techniques to directly visualize and quantify BTV infection in Culicoides cell culture by immune infrared fluorescence staining. These techniques will facilitate the use of the Culicoides cell lines in research questions and for isolation and quantification of BTV from biological samples.

Technical Abstract: Bluetongue virus (BTV) is transmitted to sheep, cattle and other ruminants by Culicoides spp. of biting midges. Cell lines have been developed from C. sonorensis; however, techniques for directly detecting and quantifying virus in these insect cells are lacking. In situ immune infrared fluorescent staining techniques were developed to visualize and quantify BTV infection in Culicoides cell culture by both an endpoint titration and an agarose overlay fluorescent focus assay. Insect cell cultures infected with BTV were fixed, permeabilized and reacted with virus-specific monoclonal antibody and fluorescent-labeled secondary antibody. Virus replication in the infected cells was visualized and quantified by measuring fluorescence with an infrared imager. The sensitivity of virus detection in the insect cell culture using these techniques was comparable to detection by standard techniques in vertebrate cell culture.